Fluorescent histochemical localization of neutral endopeptidase-24.11 (enkephalinase) in the rat brainstem

Abstract

Characterization of the distribution of the peptide-degrading enzyme neutral endopeptidase-24.11 (E.C. 3.4.24.11; NEP; enkephalinase) in the rat brainstem was examined by means of a unique fluorescent histochemical method. Enzyme staining was completely blocked by three potent NEP inhibitors (thiorphan, phosphoramidon, and JHF-26) at a concentration of 50 nM, supporting the specificity of this method to visualize sites of NEP activity selectively. At all levels of the brainstem, NEP was localized to cell bodies, cell processes or terminal-like fields and was localized to more than 90 distinct nuclei or subnuclei. In the mesencephalon these included the central gray, cuneiform n., dorsal and lateral tegmental n., inferior colliculus, interpeduncular n., lateral and medial geniculate n., central linear raphe n., mesencephalic n. of the trigeminal nerve, mammillary nuclei, occulomotor n., red n., superior colliculus, ventral n. of the lateral lemniscus, substantia nigra-ventral tegmental area, and the zona incerta. In the pons, NEP staining was restricted to fewer regions or nuclei, including the dorsal and ventral cochlear n., facial n., motor trigeminal n., principal sensory trigeminal n., parabrachial nuclei, pontine n., the oral and caudal pontine reticular n., pontine olivary nuclei, several pontine tegmental nuclei, pontine raphe nuclei, and the trapezoid n. In the cerebellum, staining was localized largely to the granule cell layer of the cerebellar cortex. Scattered staining was observed in the molecular cell layer. The medulla contained extensive NEP staining localized to nuclei that included the ambiguous n., dorsal motor n. of the vagus, hypoglossal n., inferior olivary n., prepositus hypoglossus n., solitary tract n., nuclei of the spinal tract of the trigeminal n., and the lateral, medial, and superior vestibular nuclei. Nuclei of the medullary reticular formation that were also richly stained for NEP included the raphe magnus n., raphe obscurus n., raphe pallidus n., dorsal, lateral, and ventral reticular nuclei of the medulla, and the gigantocellular, lateral paragigantocellular, linear, paramedian and parvicellular reticular nuclei. The widespread distribution of NEP in the brainstem suggests the existence of a number of functional systems, including the pathways involved in the mechanisms of pain and analgesia, which are potential targets of NEP inhibitors. In most regions, the distribution of NEP closely overlapped with that reported for the enkephalins, and showed a more restricted overlap with the reported distribution of substance P.